U.S. patent number 3,929,580 [Application Number 05/501,587] was granted by the patent office on 1975-12-30 for creatine phosphokinase test indicator.
This patent grant is currently assigned to American Cyanamid Company. Invention is credited to Peter Salvatore Forgione, Howard M. Robbins.
United States Patent |
3,929,580 |
Forgione , et al. |
December 30, 1975 |
Creatine phosphokinase test indicator
Abstract
A diagnostic test indicator is prepared for the detection and
determination of the concentration of creatine phosphokinase in
sera comprising three superimposed layers the upper of which has
contained therein the dried residue resulting from the impregnation
thereof with 1. creatine phosphate and 2. adenine diphosphate, the
middle of which has contained therein the dried residue resulting
from the impregnation thereof with 3. glucose, 4. hexokinase, 5.
triphosphopyridine nucleotide, 6. glucose-6-phosphate
dehydrogenase, 7. magnesium ion and 8. d-maltose and the lower of
which has contained therein 9. a tetrazolium salt and 10. phenazine
methosulfate.
Inventors: |
Forgione; Peter Salvatore
(Stamford, CT), Robbins; Howard M. (Chicago, IL) |
Assignee: |
American Cyanamid Company
(Stamford, CT)
|
Family
ID: |
23994178 |
Appl.
No.: |
05/501,587 |
Filed: |
August 29, 1974 |
Current U.S.
Class: |
435/14; 435/15;
435/17; 435/26; 435/805; 422/423 |
Current CPC
Class: |
C12Q
1/50 (20130101); Y10S 435/805 (20130101) |
Current International
Class: |
C12Q
1/50 (20060101); G01N 031/14 () |
Field of
Search: |
;195/99,63,13.5R,68
;23/253TP |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Naff; David M.
Attorney, Agent or Firm: Van Riet; Frank M.
Claims
We claim:
1. A diagnostic test indicator for detection and concentration
determination of creatine phosphokinase in sera comprising, in
superimposed relationship,
A. an upper layer comprising a bibulous carrier material which has
contained therein, at a pH of from about 7.2 to about 7.6, the
dried residue resulting from the impregnation thereof with
1. creatine phosphate and
2. adenine diphosphate,
B. a middle layer comprising a cellulosic, bibulous material which
has contained therein, at a pH of from about 7.1 to about 7.6, the
dried residue resulting from the impregnation thereof with
3. glucose,
4. hexokinase
5. triphosphopyridine nucleotide,
6. glucose-6-phosphate dehydrogenase,
7. magnesium ion and
8. d-maltose and
C. a lower layer comprising a cast, water-soluble polymeric
material which has contained therein, at a pH of from about 3.0 to
about 6.0,
9. a tetrazolium salt and
10. phenazine methosulfate.
2. A diagnostic test indicator according to claim 1 wherein said
(9) is 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium
chloride.
3. a diagnostic test indicator according to claim 1 wherein said
(9) is nitroblue tetrazolium.
4. A diagnostic test indicator according to claim 1 wherein said
(A) also contains a buffer.
5. A diagnostic test indicator according to claim 1 wherein said
(B) also contains a buffer.
6. A diagnostic test indicator according to claim 4 wherein said
buffer is tris buffer.
7. A diagnostic test indicator according to claim 5 wherein said
buffer is tris buffer.
8. A diagnostic test indicator according to claim 1 wherein said
(B) and (C) are adhered to one another.
9. A diagnostic test indicator according to claim 1 wherein said
(A) is a cellulose acetate semi-permeable membrane.
10. A diagnostic test indicator according to claim 1 wherein said
(B) is paper.
11. A diagnostic test indicator according to claim 1 wherein said
(C) is a film of methyl cellulose.
12. A process for the preparation of the diagnostic test indicator
of claim 1 which comprises impregnating a first bibulous material
with an aqueous solution of said (1) and (2), thereafter drying the
thus impregnated first material, impregnating a second bibulous
material with an aqueous solution of said (3), (4), (5), (6), (7),
and (8), thereafter drying the thus impregnated second material,
casting a film from a solution containing said (9) and (10) and
combining the thus prepared first and second dried materials and
cast film in superimposed relationship.
13. A process according to claim 12 wherein said (9) is
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium
chloride.
14. A process according to claim 12 wherein said (9) is nitroblue
tetrazolium.
15. A process according to claim 12 wherein said aqueous solutions
contain a buffer.
16. A process according to claim 12 wherein said second dried
material and said cast film are adhered to one another.
17. A process according to claim 12 wherein said first bibulous
material is a cellulose acetate semi-permeable membrane.
18. A process according to claim 12 wherein said second bibulous
material is paper.
19. A process according to claim 12 wherein said film is cast from
a methyl cellulose solution.
20. A process according to claim 15 wherein said buffer is tris
buffer.
Description
BACKGROUND OF THE INVENTION
The use of diagnostic test in the clinical testing of patients has
become increasingly common in recent years. Many of these tests
employ the use of test papers wherein the individual conducting the
test merely contacts a reactive paper strip with a suspect
material, usually a body fluid, and observes the color change or
color intensity thereof in order to determine whether or not a
particular effect is achieved. Test strips or cards of this type
have been devised for glucose determination and treponemal
diseases, for example.
Most tests for the detection of the concentration of creatine
phosphokinase in body fluids have, until now, consisted of
extremely complex liquid or tablet systems whereby cuvettes,
devices such as spectrophotometers, ultraviolet light, timing
instruments, constant temperature devices and individual
standardizations etc. or intricate test plates (U.S. Pat. No.
3,663,374) must be used. There has therefore existed, for a
substantial period of time, the need for a simple testing mechanism
for the determination of the concentration of creatine
phosphokinase, especially in sera, which long-felt need is
satisfied by the instant invention more fully discussed
hereinbelow.
SUMMARY
As mentioned briefly above, I have now discovered a novel test
means for the determination of the concentration of creatine
phosphokinase in body fluids. My test indicator is useful for the
qualitative detection and quantitative determination of creatine
phosphokinase and comprises reagent compositions incorporated
within three superimposed layers.
The quantitative determination of creatine phosphokinase is
extremely important in the detection of heart diseases in that the
concentration of creatine phosphokinase in the blood is elevated
noticeably over its normal concentration when diseases of the heart
exist. The early detection of an abnormal rise in creatine
phosphokinase concentration can therefore obviously lead to a more
accurate and rapid diagnosis of heart maladies and consequently,
the more rapid treatment thereof.
Because early diagnosis of abnormal heart conditions is so
important, a test for the detection of variables in the
concentration of creatine phosphokinase in the blood must be rapid
and simple enough for the clinician to carry out but accurate
enough to enable the diagnosis to be made without extreme chances
of error or false readings. Such a mechanism is represented by the
novel test indicators of the present invention. Utilizing my novel
system, no instrumentation is necessary and no mixing or
reconstitution of reagents is needed. Testing can therefore be
conducted at home or in a doctor's office without any special
equipment.
In the normal, generally used, solution testing of sera for
creatine phosphokinase, reagent compatibility is not a problem.
However, incorporation of the solution reagents together in a
specific system, as herein, presents serious instability problems
even during short term storage thereof since the color indicator, a
tetrazolium salt-phenazine methosulfate mixture, is unstable at the
pH at which the test must be carried out. Furthermore, many of the
reagents used are incompatible with one another and therefore
cannot be used in intimate contact with one another as in known
test indicators. I have overcome these serious problems by
incorporating the reagent components in individual strata, each of
which is at its own stable pH and each of which contain compatible
reactants, which strata are either separated by barrier adhesive
layers permeable only to various reaction products or are composed
of materials in the form of films which contain reagents and keep
then apart from other reagents with which that are
incompatible.
DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS AND
BEST MODE OF OPERATION
The novel diagnostic test indicators for the determination of the
concentration of creatine phosphokinase, hereinafter sometimes
referred to as CPK, of the present invention comprise three layers,
the upper one, or that with which the sera is originally contacted,
of which is composed of a bibulous carrier material such as a
cellulosic paper, a cellulose acetate semi-permeable membrane etc.
which contains therein the dried residue resulting from the
impregnation thereof with various reagent materials.
In the preparation of my novel indicators, the upper layer is
impregnated with an aqueous solution of a creatine phosphate (CP)
and adenine diphosphate (ADP), in a buffer. Both phosphates are
incorporated into the upper layer as solutions in water. Creatine
phosphate is employed in amounts ranging from about 0.125 part to
about 15.0 parts, based on 100 parts of water used while adenine
diphosphate is employed in amounts ranging from about 0.0125 part
to about 2.0 parts, based on 100 parts of water used. Mixtures of
the creatine phosphate and the adenine diphosphate may be used, if
desired, and may be incorporated into the upper layer as a mixture
or individually as long as the concentrations thereof fall within
the above ranges.
The (CP) and (ADP) are applied to the material forming the upper
layer of the indicator only because of incompatibilities of these
phosphates with components in the middle layer. The resultant dried
residue in the layer should preferably have a pH ranging from about
7.2 to about 7.6, which can be accomplished by buffering the
solution of these components with a buffer such that the pH of the
buffer solution ranges from about 7.4 to about 7.7. Impregnation
and drying of the bibulous material or semi-permeable membrane with
such a solution results in the desired pH range on the resultant
bibulous material.
Examples of suitable buffers include carbonate buffer, phosphate
buffer, phthalate buffer, "tris" buffer, glycine, citrate-phosphate
buffer, borate-succinate buffer and the like. The preferred buffer
is "tris" buffer in from about 0.1 to about 1.0 M
concentrations.
The middle or second layer of my novel test indicators is composed
of a cellulosic bibulous material such as a cellulosic paper etc.,
which is impregnated with six ingredients which may be incorporated
therein from a series of single aqueous or solvent solutions or as
solutions of two or more ingredients. Aqueous "tris" buffered
solutions are preferred. The first of these essential ingredients
is glucose which is used in amounts ranging from about 0.1 part to
about 2.0 parts, preferably from about 0.7 part to about 0.9 part,
based on 100 parts of water used.
The second component in the middle layer of my novel test
indicators is hexokinase, an enzyme known to be effective in
creatine phosphokinase testing. This enzyme is incorporated in
amounts ranging from about 15,000 I.U. to about 40,000 I.U.,
preferably from about 25,000 I.U. to about 35,000 I.U., based on
100 parts of water used.
Triphosphopyridine nucleotide is the third essential ingredient
impregnated into the middle layer. This additive is used in
concentrations ranging from about 400.0 parts to about 800.0 parts,
preferably from about 600.0 parts to about 700.0 parts, based on
100 parts of water used.
The fourth critical constituent in the middle layer of my novel
creatine phosphokinase test indicators is glycose-6-phosphate
dehydrogenase, another enzyme which catalyzes the reaction which
occurs between the reactants and reaction products in the
indicator. This enzyme is present in amounts ranging from about
3,000 I.U. to about 7,000 I.U., preferably from about 4,000 I.U. to
about 6,000 I.U., based on 100 parts of water used.
The fifth component which is incorporated into the middle layer is
a magnesium ion which functions as a catalyst to initiate the
reactions which occur therein. This magnesium ion may be present as
a water-soluble magnesium salt such as magnesium chloride,
magnesium nitrate, magnesium citrate, magnesium sulfate, magnesium
acetate, magnesium bromide, etc. in amounts ranging from about
0.001 M to about 0.1 M, preferably from about 0.005 M to about 0.01
M.
The d-maltose, which comprises the last essential material
impregnated into the middle layer i.e. bibulous, cellulosic
carrier, functions as a stabilizer for the enzymes present therein
so that storage of the final indicator does not inactivate the
enzymes thereby rendering the indicator useless. The d-maltose is
present in stabilizing amounts which generally range between about
10.0 and 35.0 parts, based on 100 parts of water used.
As mentioned above, these six ingredients can be incorporated into
the bibulous carrier from individual aqueous solutions thereof with
drying of the impregnated material being effected between each
incorporation, or as mixed solutions, the ultimate impregnated
material being thereafter dried and ready for incorporation into
the final structure. The active ingredients must be applied to the
bibulous material so that the resultant dried residue has a pH
ranging from about 7.1 to about 7.6. This can be accomplished, by
incorporating into the impregnating solution(s) a sufficient amount
of any of the above suitable buffers so as to provide a solution of
a pH of from about 7.2 to about 7.7.
The third or bottom layer of my novel creatine phosphokinase test
indicators is composed of a cast film of a water-soluble polymeric
material such as hydroxyethyl cellulose, carboxymethyl cellulose,
maleic acid-methyl vinyl ether copolymers, methyl cellulose and the
like (film thickness about 0.03 inch) containing from about 0.1
part to about 0.3 part, preferably from about 0.1 part to about 0.2
part, based on 100 parts of water used, of a tetrazolium salt
indicator and from about 0.002 part to about 0.01 part, preferably
from about 0.003 part to about 0.006 part, based on 100 parts of
water used, of phenazine methosulfate.
This bottom layer is produced by casting a film from a solution of
the polymer and additives and is capable of imparting to the area
of the bottom layer with which the reduced triphosphopyridine
nucleotide, produced upon reaction of the ingredients in the middle
layer, comes into contact, a color of such varying intensity as to
be representative of the concentration of the CPK in the test sera
which is added to the upper bibulous material. These tetrazolium
dyes are also well known and generally have the formula ##EQU1##
wherein R.sub.1, R.sub.2 and R.sub.3 are the same or different aryl
or substituted aryl radicals and x is an anion such as a halide
etc.
Examples of useful salts include
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride
(INT); nitroblue tetrazolium; blue tetrazolium;
2,3,5-triphenyl-2H-tetrazolium chloride and the like.
The pH of the tetrazolium salt, containing polymeric casting i.e.
the bottom layer, should range from about 3.0 to about 6.0, the
tetrazolium salt solution being adjustable to such a pH by the
addition of an acid, such as hydrochloric acid, thereto before
casting.
These salts are incorporated into the bottom layer of the indicator
in concentrations ranging from about 0.05 part to about 0.35 part,
preferably from about 0.1 part to about 0.2 part, by weight, based
on 100 parts of water used.
The case film of water-soluble polymer from which the bottom layer
is fabricated is itself water-soluble and relatively thin. As a
result, the area of the film which comes into contact with the sera
dissolves, giving the film essentially a zero thickness at that
point. In this manner, the intensity of the color is greatly
enhanced, a feature which makes the instant indicators more
commercially attractive.
The mechanism by which the instant test indicators function is not
completely understood however, although not wishing to be bound by
any particular theory, it is believed that the reaction occurs
thusly:
creatine phosphokinase (1) Creatine phosphate + adenine .fwdarw.
creatine + adenine diphosphate triphosphate hexokinase (2) Glucose
+ adenine .fwdarw. glucose-6-phosphate + adenine triphosphate
diphosphate glucose-6-phosphate (3) Glucose-6-phosphate +
triphosphopyridine .fwdarw. nucleotide dehydrogenase
gluconate-6-phosphate + NADPH (reduced triphosphopyridine
nucleotide) phenazine (4) NADPH + Tetrazolium salt .fwdarw.
triphosphopyridine + formazan (colorless) methosulfate nucleotide
(red)
The impregnation times during any of the impregnations discussed
above for the upper and middle layers are not critical as long as
the bibulous material is fully saturated by the solution with which
it is contacted. Generally, submersion of the bibulous material in
the solution for from about 5 to about 15 minutes is sufficient in
all cases.
It can be seen therefore, that once sera is added to the test
indicator, the creatine phosphokinase therein causes a results
which subsequently result in the reduction of the tetrazolium salt
and the formation of a colored indicator, the intensity of which is
directly proportional to the concentration of the CPK in the sera.
The clinician then merely compares the color which results to a
standard color chart to ascertain the CPK concentration of the sera
being tested.
A suitable non-ionic wetting agent, any of those which are
well-known to the skilled artisan also may be incorporated into the
upper, middle or lower layers of the indicator to cause uniform
dispersion of the ingredients of the test indicator throughout each
layer. For instance, I may utilize the fatty alkanolamides i.e. the
alkanolamine reaction products with fatty acids such as lauric acid
or stripped coconut fatty acid, suitable alkanolamines being
diethanolamine, monoethanolamine, monoisopropanolamine, and the
like; the ethylene oxide derived material, i.e. those derived from
the reaction of ethylene oxide with alkylphenols wherein the alkyl
group is octyl, nonyl or higher, long chain fatty alcohols such as
tridecyl alcohol, lanolin, lecithin alcohol etc., long chain fatty
acids such as tall oil, oleic acid, abietic acid etc., long chain
fatty mercaptans, long chain fatty amines, polyoxypropylene glycol,
fatty sorbitan ester; sugaar esters i.e. the alcoholysis reaction
products of the methyl ester of a fatty acid and sucrose or
raffinose; polysorbitol; polyvinyl alcohol; methyl cellulose;
ethoxylated phenol/formaldehyde resins and the like. These
materials may be added along with each component of the indicator,
if applied singly, or in admixture with component mixtures if
applied as such in amounts of 0.01 part to 1.0 part of wetting
agent per 100 parts of solution used.
The upper, middle and lower layers, after drying, are then arranged
in superimposed relationship in the fabrication of the final test
indicators of the present invention. It is preferred that the lower
and middle layers be attached to one another by applying an organic
solvent soluble adhesive, permeable to the NADPH and the buffers
used in the middle layer, to one side of one or both layers and
pressing them together. Such a procedure is not, however, required.
Suitable adhesives include cellulose acetate, cellulose
phthalate-acetate mixture, polyvinyl chloride, polyvinyl
alcohol-acetate mixtures and the like. Excellent results have been
achieved by precoating one or both layers with a first layer of
adhesive and drying before applying a second adhesive layer to
effect final adhesion of the two materials together. Adhesive
thickness of from about 0.01 to about 0.02 inch is
satisfactory.
The main criteria for the adhesive system employed, because it
covers the entire surface of either or both of the middle and lower
layers, is that the produced NADPH in buffer therein, can pass
therethrough and react with the reactants in the lower,
water-soluble layer.
The combined lower and middle layers are then placed below the
bottom of the upper layer in direct contact therewith, with or
without the addition of an adhesive therebetween. No adhesive is
the preferred manner. The upper layer may be held in direct contact
with the middle layer by pressure such as by placing the entire
three layer indicator on a self-supporting, transparent backing
medium, which preferably cold flows on pressure and is of a larger
dimension than the indicator, and holding the indicator in place on
said medium by placing a capping section of the same material and
same dimensions atop the entire structure. The capping section
possesses a perforation therein of smaller dimension than the
indicator and thereby exposes the center area of the upper layer of
the indicator so that sera may be added thereto. The cappinng
section is adhered around the periphery of the indicator to the
self-supporting medium by applying pressure thereto. The indicator
is then read through the transparent backing medium.
The colors of the indicators of the present invention generally
range from deep red which is indicative of a high CPK level in the
test serum to pink which indicates a normal CPK level. Nitroblue
tetrazolium and blue tetrazolium result in, as expected, dark blue
indicator colors upon contact with sera high in creatine
phosphokinase.
The above concentrations expressed in connection with the
components which may be incorporated into my novel indicators are
set forth as to the solutions of these components which are
impregnated into the bibulous carriers or formed into castings only
and are not meant to specify the amount of each component which
eventually remains on the bibulous carrier or is present in the
final casting. That is to say, saturation of the bibulous carrier
with a specific concentration of a specific component in solution
or casting a specific polymer solution will not unequivocally
incorporate into the bibulous carrier or the resultant casting the
identical amount or percentage of component present in the
solution. I have found, however, that the above concentrations of
solution are generally sufficient so as to produce functional
castings and so as to incorporate sufficient amounts of each
component into the bibulous material upon saturation therewith to
produce a functional test indicator, the absorptive capabilities of
the bibulous material being characteristic of materials generally
used for this purpose.
The following examples are set forth for purposes of illustration
only and are not to be construed as limitations on the present
invention except as set forth in the appended claims. All parts and
percentages are by weight unless otherwise specified.
EXAMPLE 1
Formation of Upper Layer
An aqueous solution is prepared of 0.12 part of creatine phosphate
and .0185 part of adenine diphosphate in 1.0 part of 0.1 M "tris"
buffer at a pH of 7.5. A circular piece (2 inch diameter) of a
commercially available cellulose acetate membrane filter (Metricel
GA -- 3 -- Gelman Co.) is immersed in the solution for 5 minutes,
removed and vacuum dried in the dark at room temperature.
Formation of Middle Layer
An aqueous solution is formed of 0.054 part of glucose, 0.045 part
of triphosphopyridine nucleotide, 2.0 parts of d-maltose, 2,100
I.U. of hexokinase suspension and 330 I.U. of glucose-6-phosphate
dehydrogenase in 7.0 parts of 0.1 M "tris" buffer, pH 7.5,
containing 10.sup.-.sup.2 M magnesium chloride. A circular piece (2
inch diameter) of Whatman No. 42 filter paper is immersed in the
solution for 6 minutes, removed, blotted of excess liquid and dried
in the dark under vacuum at room temperature.
Formation of Lower Layer
A 0.03 inch water-soluble thick film is cast on a glass surface and
in total darkness from a solution of 5.0 parts of methyl cellulose,
0.2 part of
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride
and 0.005 part of phenazine methosulfate, acidified with acid to a
pH of 4.0.
Fabrication of Test Indicator
The lower and middle layers are cut into 6 mm diameter circular
sections and are adhered together with a thin coating of bonding
agent by painting a solution containing 10.0% cellulose acetate in
ethyl acetate on the middle layer, partially drying the coating and
compressing the two layers under slight pressure. The combined
layers are then placed below the upper layer with the middle layer
side up to form the indicator assembly. A stiff section (10 mm
diameter) of a commercially available transparent plastic with cold
flow properties with a 5.8 mm diameter hole therein is then placed
over the assembly with the hole directly atop the upper layer of
test papers. A continuous section (10 mm diameter) of similar
material is then placed directly below the lower layer of the
assembly as a backing medium and the edges of the overlay and the
backing medium are then bonded together by pressure.
A drop of suspect sera is then placed in the hole atop the final
assembly in contact with the upper layer. After 15 minutes a dark
red spot is observed on the underside of the lower layer indicative
of an abnormal amount of creatine phosphokinase in the tested
sera.
EXAMPLE 2
The procedure of Example 1 is again followed except that the upper
membrane filter is replaced by Whatman No. 42 filter paper. Similar
results are observed when the resultant indicator is contacted with
the suspect sera.
EXAMPLE 3
The procedure of Example 1 is again followed except that the
magnesium salt is magnesium acetate. Again a sensitive creatine
phosphokinase indicator is produced.
EXAMPLE 4
Following the procedure of Example 1, a successful testing for
creatine phosphokinase is made on test sera, the lower layer being
cast from a solution of hydroxyethyl cellulose instead of the
material used therein.
EXAMPLE 5
The procedure of Example 4 is again followed except that the lower
layer is cast from a maleic acid-methyl vinyl ether copolymer
(80/20). Substantially identical results are achieved.
EXAMPLE 6
The procedure of Example 1 is again followed except the tetrazolium
chloride is replaced by a similar amount of nitroblue tetrazolium.
An excellent creatine phosphokinase indicator is produced. A dark
blue color results when testing the suspect sera.
EXAMPLE 7
When following the procedure of Example 6 except the blue
tetrazolium is used, substantially identical results are observed.
The color is dark blue.
EXAMPLE 8
Again following the procedure of Example 6, an excellent indicator
results when 2, 3, 5-triphenyl-2H-tetrazolium chloride is employed
as the tetrazolium salt. The color is very deep pink.
EXAMPLE 9
The procedure of Example 1 is again conducted except that the
adhesive bonding between the middle and lower layers is eliminated.
Similar results are achieved.
EXAMPLE 10
The procedure of Example 1 is again followed except that
polyethylene (23) lauryl ether is incorporated into each solution
from which the layers are formed in order to increase the
dispersion of the ingredients over the surface of the layers. Again
a very successful test indicator is formed.
* * * * *